三个葡萄WRKYs基因的克隆及表达特性分析

Gene Cloning and Expression Analysis of Three WRKYs in Vitis vinifera L.

以葡萄抗性品种‘左优红’组培苗为材料,利用同源克隆法克隆VvWRKY13、VvWRKY45和VvWRKY71,测序结果显示,VvWRKY13扩增片段大小为681bp,编码226个氨基酸;VvWRKY45扩增片段大小为549bp,编码182个氨基酸;VvWRKY71扩增片段大小为936bp,编码311个氨基酸序列。利用生物信息学分析VvWRKY13、VvWRKY45和VvWRKY71序列显示,这3个蛋白均含有保守的WRKY结构域;分子量分别为25.7、20.8和34.8kDa,pI分别为9.08、9.41和6.94。实时定量PCR结果表明,3个WRKYs均在叶片中表达量最高。逆境相关的信号物质,如水杨酸、脱落酸、茉莉酸和一氧化氮以及高盐、低温和渗透等逆境胁迫均可诱导VvWRKY13、VvWRKY45和VvWRKY71的表达。推测3个基因均参与了葡萄抵御逆境胁迫的过程。

WRKY is an important transcription factor that can regulate the expression of downstream stress resistance genes, it can be widely involved in plant biotic and abiotic stress resistance. Using homology cloning method, the full-length cDNA of VvWRKY13, VvWRKY45 and VvWRKYT1 was cloned from leaves of Vitis vin- ifera cultivar ＇Zuoyouhong＇ tissue culture seedling. Bioinformatic analysis indicated that VvWRKY13, VvWRKY45 and VvWRKY71 separately consisted of 681,549, 936 nucleotides encoding 226, 182, 311 amino acid with molecular weight 25.7, 20.8, 34.8 kDa, isoelectric point 9.08, 9.41 and 6.94, respectively. Three VvWRKYs all possessed one conserved WRKY domain. Real-time PCR analysis showed that three VvWRKYs were expressed in all tested tissues, with the highest expression in leaves. VvWRKY13, VvWRKY45 and VvWRKY71 was significantly induced by stress-related signal substances such as salicylic acid （SA）, abscisic acid （ABA）, jasmonate acid （JA）, nitric oxide （NO）. Moreover, three VvWRKYs were accumulated in response to salt stress, cold stress and osmotic stress. It suggested these three VvWRKYs were involved in resistance to various stresses in grape.